The present invention relates to a method of detection of DNA and protein and of DNA base sequencing determination and to an apparatus therefor.
It relates more particularly to a fluorescence detection type gel electrophoresis apparatus.
For DNA base sequencing by electrophoresis gel separation, a radioisotope label has been used as a label for a DNA fragment. Due to the inconvenience of this method, however, a method of using a fluorophore label has come to be increasingly employed. See, for example, U.S. patent application Ser. No. 07/506,986 (U.S. Pat. No. 5,062,942) and Bio/Technology Vol. 6, July 1988, pp.816-821. As an excitation light source, this method uses an argon laser with an output of 20 to 50 mw and a wavelength of 488 nm or 515 nm to detect a DNA fragment of 10.sup.-16 mole/band to 2.times.10.sup.-18 mole/band. As fluorophores, the method has used FITC (fluorescein isothiocyanate with a maximum emission wavelength of 515 nm), SF (succinyl fluorescein with a maximum emission wavelength of 510 nm to 540 nm), TRITC (tetrarhodamine isothiocyanate with a maximum emission wavelength of 580 nm) and Texas Red (sulforhodamine 101 with a maximum emission wavelength of 615 nm).
Normally, electrophoresis is performed with a polyacrylamide gel plate which is provided between two glass plates. In recent years, a capillary gel electrophoresis method has been developed, where gel is formed in a capillary. Use of a capillary having a smaller diameter increases the surface area per volume of gel; this feature facilities the dissipation of Joule heat, permitting application of high voltage. A high-speed electrophoresis separation is provided by the capillary gel electrophoresis method.
The first examples of the capillary gel electrophoresis method in the prior art are described in Nucleic Acids Research, Vol. 18pp. 1415 to 1419 (1990), Journal of Chromatography, Vol. 516, pp. 61-67 (1990), and Science, Vol. 242, pp. 562-564 (1988). Another method of using one migration lane for base sequence determination is disclosed in Nature, Vol. 321, pp. 674-679 (1986) and elsewhere.
The above-mentioned conventional technique, however, has disadvantages in that the sensitivity is insufficient, and the entire equipment must be made greater in size because the Ar laser is greater in size than a He-Ne laser.
In the prior art, gel electrophoresis has been used for DNA analysis including DNA sequencing. In recent years there have been growing demands for DNA sequencing such as genome analysis. At the same time, there has become available on the market a DNA sequencer which measures DNA fragments on a real-time basis by labeling them with a fluorophore, while performing gel electrophoresis separation; this device has already been put into practical use. This DNA sequencer uses a gel plate (polyacrylamide gel), which is provided with 30 to 40 migrating lanes. The gel plate is exposed to the laser beam at specified intervals from the electrophoresis starting point, and the sequencer detects the fluorescence emitted by the DNA fragments labeled with a fluorophore passing through the irradiated site.
However, the throughput of the DNA sequencer available on the market is as small as 10,000 bases to 20,000 bases/day; this has been one of the problems hindering genome analysis. To ensure high throughput, it is effective to increase the speed of electrophoresis and to provide a great number of migrating lanes, thereby increasing the number of samples to be detected at one time.
In recent years there has been developed a capillary gel electrophoresis apparatus which enables electrophoresis separation in a short time. Furthermore, a capillary array electrophoresis apparatus has been developed; it allows simultaneous measurement of many samples with an arrangement of a great many capillaries, thereby providing a high throughput.
The following capillary array electrophoresis apparatus of this type has been reported, an apparatus where an array of the capillaries placed on a flat board is secured to an X-Y table, and is exposed to a laser beam from above to detect fluorescence according to the direction of the laser beam (Nature, Vol. 359, pp. 167-169 (1992)). In this apparatus, only one capillary is exposed to the laser beam at one time, and the capillary gel is scanned mechanically at a specified speed to measure fluorescence signals from all capillaries.